High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets.

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)-induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin-induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma-thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma-thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.     

Down-regulation of the serotonin transporter in hyperreactive platelets counteracts the pro-thrombotic effect of serotonin

An elevated plasma concentration of serotonin ([5-HT]) is a common feature of cardiovascular disease often associated with enhanced platelet activation and thrombosis. Whether elevated in vivo plasma 5-HT per se represents an independent risk factor for platelet hyperreactivity or only is an epiphenomenon of cardiovascular disease is poorly understood. We examined in vitro and in vivo platelet function following a 24h elevation of plasma [5-HT] in mice. In vivo administration of 5-HT using osmotic minipumps increased plasma [5-HT] in treated mice compared to control mice instrumented with saline loaded pumps. 5-HT infusion did not increase systolic blood pressure, but markers of platelet activation including P-selectin and (PE)Jon/A staining were increased and these findings coincided with the enhanced aggregation of isolated platelets in response to type I fibrillar collagen. Tail bleeding times and the time to occlusion following chemical damage to the carotid artery were shortened in 5-HT-infused mice. 5-HT-infused mice were treated with paroxetine (Prx) to block 5-HT uptake via the serotonin transporter (SERT). Prx lowered platelet [5-HT] and attenuated platelet activation and aggregation. These results and our biochemical indices of enhanced 5-HT intracellular signaling in the platelets of 5-HT-infused mice reveal a mechanistic link between elevated plasma [5-HT], abnormal intracellular 5-HT signaling and accentuated platelet aggregation. Although a down-regulation of the serotonin transporter (SERT) on the platelet surface may counteract the pro-thrombotic influence of elevated plasma [5HT], this compensatory mechanism may fail to prevent the increased thrombotic risk caused by elevated plasma [5-HT].     

Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen-induced platelet aggregation by nitroprusside

In studies on human platelets, nitroprusside (NP) alone at 1-10 micromol/l increased platelet cyclic AMP (cAMP) by 40-70%, whereas increases in cyclic GMP (cGMP) were much larger in percentage though not in concentration terms. Collagen enhanced these increases in cAMP up to fourfold, without affecting cGMP. This effect was partly prevented by indomethacin or aspirin, indicating that platelet cyclo-oxygenase products acted synergistically with NP to increase cAMP. ADP released from the platelets by collagen tended to restrict this cAMP accumulation. Addition of 2',5'-dideoxyadenosine (DDA), an inhibitor of adenylyl cyclase, decreased both the inhibition of collagen-induced platelet aggregation by NP and the associated accumulation of cAMP without affecting cGMP, indicating that cAMP mediates part of the inhibitory effect of NP. Unlike DDA, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of guanylyl cyclase, blocked all increases in both cGMP and cAMP caused by NP, as well as the inhibition of platelet aggregation, suggesting that cAMP accumulation was secondary to that of cGMP. Human platelet cGMP-dependent protein kinase (PKG) coelectrophoresed with the purified bovine type Ibeta isoenzyme. An inhibitor of this enzyme (Rp)-beta-phenyl-1,N2-etheno-8-bromoguanosine 3',5'-cyclic-monophosphorothioate, diminished the inhibition of collagen-induced platelet aggregation by NP, but had little additional effect when DDA was present. This showed that both PKG and cAMP participate in the inhibition of collagen-induced platelet aggregation by NP. Moreover, selective activators of PKG and cAMP-dependent protein kinases had supra-additive inhibitory effects, suggesting that an optimal inhibitory effect of NP requires simultaneous activation of both enzymes.     

In vitro platelet abnormality in adenosine deaminase deficiency and severe combined immunodeficiency.

The platelets of an infant with severe combined immune deficiency and adenosine deaminase deficiency showed markedly diminished responses to ADP-induced aggregation in vitro. This abnormality was corrected by the addition of purified adenosine deaminase in vitro. Exogenous adenosine added to platelet-rich plasma caused markedly prolonged inhibition of ADP-induced aggregation. This was shown by isotopic studies to be due to slow clearance of adenosine and hence persistence of this nucleoside. Direct assay for adenosine deaminiase in plasma and platelet lysates of the patient confirmed the very low activity of this enzyme. Raised cAMP levels were demonstrated in his platelets. The deranged adenosine metabolism and raised cAMP in the platelets of this child with severe combined immunodeficiency may explain the altered response to ADP. Despite the in vitro platelet aggregation abnormality, the patient had no clinical evidence of impaired hemostasis.     

Experimental study of preventing emphysema with elastase inhibiting agents

We have observed the influence of Radix Salviae Miltiorrhizae and Ligustrazini Hydrochloridum on the elastase activity and the protective effects of the two medicines on the elastic fibers. The results showed that Radix Salviae Miltiorrhizae and Ligustrazini Hydrochloridum can inhibit the activity of porcine pancreatic elastase and human sputum elastase. These two drugs can protect arterial and pulmonary elastic fibers from the destruction of elastase. Since these traditional Chinese herbs have little toxic effect and no antigenic character, they can be used in the preventing emphysema. The study suggest that it is useful Radix Salviae Miltiorrhizae and Ligustrazini Hydrochloridum are as favourable elastase inhibiting agents, which may be used in the prevention of emphysema extensively.     

Inhibitory effect of Ginkgo biloba extract on human platelet aggregation

The effect of pure flavonoids and Gingko biloba extract (GBE) on human platelet aggregation was investigated. Most of the flavonoids and vitamin E did not affect platelet aggregation in platelet-rich plasma (PRP); however some of these flavonoids inhibited platelet aggregation in gel-filtered platelets (GFP). GBE inhibited both ADP- and collagen-induced platelet aggregation in PRP, GFP and in whole blood in a dose-dependent manner. GBE at very low concentrations inhibited whole blood aggregation induced by ADP compared with those used for PRP or GFP. Flavonoids and GBE decreased the production of TxA(2) induced by collagen and ADP in PRP. However, no correlation was observed between the inhibition of platelet aggregation and the decrease of TxA(2) synthesis. GBE and flavonoids did not affect platelet membrane fluidity. However, the incubation of PRP with GBE increased cAMP levels in platelets, which is known to inhibit platelet activation by lowering intracellular Ca2+ levels. GBE is a mixture of many compounds, including flavonoids and gingkoglides, which affect metabolism of cAMP, TxA(2) and Ca2+ in platelets. It is effective in the inhibition of platelet aggregation, both in PRP and whole blood, and thus may be potentially used as an effective oral anti-platelet therapeutic agent.     

Intraplatelet serotonin and plasma 5-hydroxyindoles in health and disease

Using a fluorometric test sensitive to serotonin (5-HT) and to other 5- hydroxyindoles (5-HIs) it was shown that platelets take up 5-HT and that the added 5-HT and the inherent test-positive material in platelet lysate can be directly measured. However, platelets do not take up 5- hydroxyindole acetic acid or the 5-HIs from the plasma. Thus, 5-HT and the other 5-HIs can be distinguished. Various methods of liberating intraplatelets 5-HT were investigated. Several anticoagulants, temperatures, and speeds of centrifugation were employed, with no effect on the results. It was found that in healthy donors, with increasing age, there was a decrease in intraplatelet 5-HT and an increase in plasma 5-HIs. The quantities of these substances were inversely related. In acute myocardial infarction, the 5-HT was normal taking age into account, but the plasma 5-HIs were increased. In postoperative patients, the platelet 5-HT was low and the plasma 5-HIs were normal. In patients with chest pain but no myocardial infarction, both platelet 5-HT and plasma 5-HIs were normal. The relevance of these findings to 5-HT metabolism and the evidence for in vivo activation of platelets is discussed.     

Relation between the Inhibition of Aggregation and the Concentration of cAMP in Human and Rat Platelets

Adenosine inhibits the aggregation of human but not of rat platelets whereas both are inhibited by prostaglandin E1 or by the pyrimido-pyrimidine compound RA233. In human platelets all three agents increase adenosine-3'-5'-cyclic monophosphate (cAMP). If the inhibition of aggregation depended on this increase, adenosine might be expected not to increase cAMP in rat platelets. Under conditions in which adenosine inhibited aggregation and increased cAMP in human platelets, adenosine caused a similar increase in cAMP in rat platelets without inhibiting their aggregation. The aggregation of rat platelets was inhibited as effectively as that of human platelets by PGE1 or RA233 at concentrations which caused greater increases in cAMP than did the highest concentrations (2.8 X 10(-4) M) of adenosine it was possible to use. When the increase of cAMP in rat platelets by PGE1 was limited to that produced by adenosine, PGE1 like adenosine failed to inhibit aggregation. Therefore, the difference in the inhibitory effectiveness of adenosine on rat and human platelets was quantitative rather than qualitative and apparently depended on the inability of adenosine to increase cAMP sufficiently in rat platelets. When cAMP had been increased by adenosine, PGE1 or RA233, the addition of ADP caused cAMP to decrease rapidly in both human and rat platelets to between +22 and -18% of control values, except that the decrease in rat platelets was to +40% after RA233 had been present for 0.5 min before ADP. The increase in cAMP produced in rat platelets by adenosine at 5 X 10(-6) to 2.8 X 10(-4) M for 3 min was associated with a small increase in aggregation velocity. It is suggested that the comparative ineffectiveness of adenosine as an inhibitor of platelet aggregation, particularly with rat but less so also with human platelets, is because, unlike PGE1 or RA233, adenosine has two opposing actions on aggregation; one being inhibition by activating adenylate cyclase and increasing cAMP, and the other being potentiation by uptake. This hypothesis accounts for the present results as well as for the earlier observation that dipyridamole which prevents the uptake of adenosine potentiates its inhibitory effect on the aggregation of human platelets.     

Inhibition of platelet aggregation by idebenone and the mechanism of the inhibition

The inhibitory effect of 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (idebenone) on platelet aggregation was studied in rat and human platelets in vitro, and the mechanism of inhibition was examined in rat platelets. Idebenone inhibited the aggregation induced by collagen and thrombin in washed platelets, and by arachidonate and ADP in platelet-rich plasma (PRP). The inhibition was more prominent in collagen- and arachidonate-induced aggregation. In collagen-induced aggregation of human platelets, idebenone was 8-fold more potent than aspirin. In addition, idebenone inhibited prostaglandin synthesis and thromboxane B2 production, and also increased the cyclic AMP content in platelets. However, the concentration of idebenone required to inhibit thromboxane B2 production was much lower than that required to increase cyclic AMP. These results indicate that idebenone inhibits platelet aggregation by inhibiting thromboxane B2 synthesis rather than by increasing cyclic AMP content.     

Sodium fluoride mimics effects of both agonists and antagonists on intact human platelets by simultaneous modulation of phospholipase C and adenylate cyclase activity

Using intact human platelets, we studied the effect of sodium fluoride (NaF) on platelet aggregation and release reaction and correlated the functional changes to intracellular events specific for either agonist- induced or antagonist-induced platelet responses. At lower concentrations, with a peak activity between 30 and 40 mmol/L, NaF induced aggregation and release of adenosine 5'-triphosphate (ATP) that was associated with increased formation of inositol phosphates, a rise in cytosolic free Ca2+, and phosphorylation of 20-kd and 40-kd proteins. At NaF concentrations greater than 40 mmol/L, aggregation and ATP release decreased dose-dependently in parallel with a decrease in Ca2+ mobilization, whereas neither inositol phosphate formation nor 40- kd protein phosphorylation was reduced. At these concentrations, NaF caused a dose-dependent transient rise in platelet cyclic adenosine 3',5'-monophosphate (cAMP) levels that was sufficient to account for the observed reduction in Ca2+ mobilization, aggregation, and ATP release. Stimulated cAMP levels started declining rapidly within 30 seconds of addition of NaF, however. Similarly, prostacyclin (PGI2)- induced cAMP accumulation was temporarily enhanced but subsequently suppressed by NaF, suggesting either stimulation of a cAMP phosphodiesterase or delayed inhibition of adenylate cyclase. Evidence for the latter was provided by the finding that NaF pretreatment of platelets resulted in partial inhibition of PGI2-stimulated cAMP formation in the presence of the cAMP phosphodiesterase inhibitor 3- isobutyl-1-methyl-xanthine (MIX). We conclude that NaF exerts a dual (stimulatory and inhibitory) effect on adenylate cyclase in intact platelets that is accompanied by simultaneous activation of a phosphoinositide-specific phospholipase C; in addition, a cAMP phosphodiesterase may be activated.     

5-Hydroxytryptamine (5-HT)-induced shape change in human platelets determined by computerized data acquisition: correlation with [125I]-iodoLSD binding at 5-HT2 receptors.

The 5-HT-induced shape change and subsequent aggregation of platelets provides a functional assay for 5-HT2 receptors. In the present study we describe a method to increase aggregometer sensitivity by digital conversion of the voltage produced by changes in light transmittance through a platelet suspension, thereby allowing accurate analyses of the primary shape-change response. The pharmacology of 5-HT-induced shape change was then compared with that of [125I]-iodoLSD binding in human platelets. 5-HT caused a dose-dependent change in platelet shape (maximum response 5 x 10(-6) M, EC50 10(-6) M). Furthermore, there was a significant correlation across a selection of drugs between IC50 values for inhibition of 5-HT-induced shape change and for inhibition of platelet binding of the 5-HT2 receptor ligand [125I]-iodoLSD. These results support the hypothesis that 5-HT-induced shape change and [125I]-iodoLSD binding in human platelets are mediated through the same receptor, and validate the methods of data acquisition described.     

A new congenital defect of platelet secretion: impaired responsiveness of the platelets to cytoplasmic free calcium

Summary . A 16-year-old boy with a bleeding disorder since infancy has a long bleeding time, normal platelet count and morphology and normal plasma factor-VIII activities. His platelets undergo normal shape change and primary aggregation in response to ADP but show defective 5-hydroxytryptamine (5-HT) secretion and aggregation in response to adrenaline, sodium arachidonate, U44069, PAF-acether, A23187 and low concentrations of collagen. Thrombin and higher concentrations of collagen produce a normal response. Secretion of β-thromboglobulin and platelet factor 4 parallels that of 5-HT. Thromboxane B₂ is produced normally in response to exogenous arachidonate and to stimulation by thrombin, collagen and A23187 in all concentrations tested. The patient's endoperoxides and thromboxane A₂ aggregate aspirin-treated platelets, though his platelets are themselves unresponsive. Cyclic AMP is present at normal concentration in the patient's unstimulated platelet-rich plasma, and PGI₂ inhibits platelet aggregation by ADP and thrombin in a normal dose-related manner. Platelet ultrastructure, 5-HT uptake and content of adenine nucleotides, platelet factor 4 and β-thromboglobulin are all within normal limits. When the patient's platelets were loaded with the fluorescent dye quin 2, which serves as an indicator of cytoplasmic free calcium ions, their responses to thrombin, whether in the presence or virtual absence of extracellular Ca2+, were entirely normal in respect of free calcium ions, secretion, shape-change and aggregation. In response to ionomycin, however, a normal increase in free calcium ions was accompanied by normal shape-change but virtually no aggregation or 5-HT secretion. The platelet calmodulin content was normal. These findings show that the defect in this patient's platelets is of utilization of cytoplasmic Caz+ for secretion and aggregation, rather than of Ca2+ uptake or mobilization of Ca2+ from intracellular storage sites. It is suggested that the most likely site of the defect is the phosphorylation of one of the proteins concerned in the secretory mechanism.     

Beta 2-glycoprotein-I (apo-H) inhibits the release reaction of human platelets during ADP-induced aggregation

In order to further characterize the modulation of the ADP-induced aggregation of gel-filtered human platelets by beta 2-glycoprotein-I (beta 2-G-I), the influence of this glycoprotein upon the serotonin (5-HT) release during aggregation was measured. The following results were obtained: beta 2-G-I completely inhibits the 5-HT release during ADP-induced platelet activation. The inhibition is correlated with the inhibition of the second wave of the ADP-induced aggregation. This effect of beta 2-G-I is not dose-dependent and appears above a threshold concentration of 0.1-0.15 mg/ml in the assay. The specificity of the beta 2-G-I interaction with the ADP-activation is supported by results obtained with collagen or thrombin as aggregating agents. In these cases neither the aggregation nor the release is influenced by the glycoprotein. In respect to the results obtained, beta 2-G-I is a potent candidate to be a modulator of ADP-induced platelet activation in vivo.     

PGE(2) reverses G(s)-mediated inhibition of platelet aggregation by interaction with EP3 receptors, but adds to non-G(s)-mediated inhibition of platelet aggregation by interaction with EP4 receptors

Prostaglandin E(2) (PGE(2)) has intriguing effects on platelet function in the presence of agents that raise cyclic adenosine 3'5'-monophosphate (cAMP). PGE(2) reverses inhibition of platelet aggregation by agents that stimulate cAMP production via a G(s)-linked receptor, but adds to the inhibition of platelet function brought about by agents that raise cAMP through other mechanisms. Here, we used the EP receptor antagonists DG-041 (which acts at the EP3 receptor) and ONO-AE3-208 (which acts at the EP4 receptor) to investigate the role of these receptors in mediating these effects of PGE(2). Platelet aggregation was measured in platelet-rich plasma obtained from healthy volunteers in response to adenosine diphosphate (ADP) using single platelet counting. The effects of a range of concentrations of PGE(2) were determined in the presence of (1) the prostacyclin mimetic iloprost, which operates through G(s)-linked IP receptors, (2) the cAMP PDE inhibitor DN9693 and (3) the direct-acting adenylate cyclase stimulator forskolin. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation was also determined as a measure of cAMP. PGE(2) reversed the inhibition of aggregation brought about by iloprost; this was prevented in the presence of the EP3 antagonist DG-041, indicating that this effect of PGE(2) is mediated via the EP3 receptor. In contrast, PGE(2) added to the inhibition of aggregation brought about by DN9693 or forskolin; this was reversed by the EP4 antagonist ONO-AE3-208, indicating that this effect of PGE(2) is mediated via the EP4 receptor. Effects on aggregation were accompanied by corresponding changes in VASP phosphorylation. The dominant role of EP3 receptors circumstances where cAMP is increased through a Gs-linked mechanism may be relevant to the situation in?vivo where platelets are maintained in an inactive state through constant exposure to prostacyclin, and thus the main effect of PGE(2) may be prothrombotic. If so, the results described here further support the potential use of an EP3 receptor antagonist in the control of atherothrombosis.     

cAMP is an important messenger for ADP-induced platelet aggregation

In rat platelets, basal cAMP levels were not changed upon stimulation with ADP and it was concluded that cAMP is not an important messenger for ADP-induced aggregation (Savi et al., Blood Coagul Fibrinolysis, 1996; 7: 249-52). In the present study, the effects of prostaglandin E(1) (PGE(1)) and ADP on human platelet aggregation, cAMP generation and VASP phosphorylation were studied. Phosphorylation of the protein kinase A (PKA) substrate VASP and inhibition of platelet aggregation by PGE(1) occurred without measurable changes in cellular cAMP levels. In addition, a marked inhibition of basal VASP phosphorylation by ADP was observed. It is concluded that cAMP determinations do not necessarily detect a possible activation or inhibition of the cAMPPKA pathway in platelets. Thus, cAMP might well be an important second messenger for ADP-induced platelet aggregation.     

Effects of nicotine on platelet function

In non-smokers, only high concentrations of nicotine (10 mM) caused platelet aggregation in platelet-rich plasma and release of 5-hydroxytryptamine (5-HT). Both responses to ADP and 5-HT were enhanced at 1 and 10 mM nicotine while they were inhibited to collagen, ristocetin, adrenaline and arachidonic acid. Lower concentrations of nicotine had no effect with any agent other than 5-HT, with which a variable enhancement of 5-HT-induced aggregation was observed. Uptake of 14C-5-HT was inhibited by nicotine at 100 microM or higher while platelet factor 3 availability was unaffected. Thus it is unlikely that direct effects of nicotine on platelets are responsible for smoking-related changes in platelet reactivity.     

cAMP is an important messenger for ADP-induced platelet aggregation

In rat platelets, basal cAMP levels were not changed upon stimulation with ADP and it was concluded that cAMP is not an important messenger for ADP-induced aggregation (Savi et al., Blood Coagul Fibrinolysis, 1996; 7: 249-52). In the present study, the effects of prostaglandin E1 (PGE1) and ADP on human platelet aggregation, cAMP generation and VASP phosphorylation were studied. Phosphorylation of the protein kinase A (PKA) substrate VASP and inhibition of platelet aggregation by PGE1 occurred without measurable changes in cellular cAMP levels. In addition, a marked inhibition of basal VASP phosphorylation by ADP was observed. It is concluded that cAMP determinations do not necessarily detect a possible activation or inhibition of the cAMPPKA pathway in platelets. Thus, cAMP might well be an important second messenger for ADP-induced platelet aggregation.     

丹参对暴发性肝衰竭大鼠肝损伤及肝再生的影响

目的：探讨丹参对暴发性肝衰竭（FHF）大鼠肝损伤及肝再生的影响。方法：Wistar大鼠随机分为4组：正常对照组、FHF组、丹参组、促肝细胞生长素（PHGF）组。FHF、丹参组和PHGF组动物模型采用TAA皮下注射,剂量按每kg体重600mg,2次,每次间隔24h,复制FHF动物模型。丹参组和PHGF组动物除皮下注射TAA外,于实验前3天至实验结束分别于皮下注射丹参注射液1ml/100g和PHGF1ml/100g,正常对照组和FHF组大鼠同时皮下注射生理盐水1ml/100g。FHF组、丹参组和PHGF组,于第2次注射TAA后24h,随机取大鼠各8只,腹主动脉取血测肝功能。迅速取肝组织,用10%甲醛液固定,制成石蜡切片,检测肝细胞有丝分裂指数（MI）和增殖细胞核抗原（PCNA）。结果：丹参具有降低FHF大鼠ALT、AST及TBil,显著提高肝细胞MI和PCNA的作用（P〈0.05,P〈0.01）。结论：丹参具有改善FHF大鼠肝功能和促进肝细胞增殖及再生的作用。     

Compartmentalisation of cAMP-dependent signalling in blood platelets: The role of lipid rafts and actin polymerisation

Abstract

Prostacyclin (PGI₂) inhibits blood platelets through the activation of membrane adenylyl cyclases (ACs) and cyclic adenosine 3',5'-monophosphate (cAMP)-mediated signalling. However, the molecular mechanism controlling cAMP signalling in blood platelet remains unclear, and in particular how individual isoforms of AC and protein kinase A (PKA) are coordinated to target distinct substrates in order to modulate platelet activation. In this study, we demonstrate that lipid rafts and the actin cytoskeleton may play a key role in regulating platelet responses to cAMP downstream of PGI₂. Disruption of lipid rafts with methyl-beta-cyclodextrin (MβCD) increased platelet sensitivity to PGI₂ and forskolin, a direct AC cyclase activator, resulting in greater inhibition of collagen-stimulated platelet aggregation. In contrast, platelet inhibition by the direct activator of PKA, 8-CPT-6-Phe-cAMP was unaffected by MβCD treatment. Consistent with the functional data, lipid raft disruption increased PGI₂-stimulated cAMP formation and proximal PKA-mediated signalling events. Platelet inhibition, cAMP formation and phosphorylation of PKA substrates in response to PGI₂ were also increased in the presence of cytochalasin D, indicating a role for actin cytoskeleton in signalling in response to PGI₂. A potential role for lipid rafts in cAMP signalling is strengthened by our finding that a pool of ACV/VI and PKA was partitioned into lipid rafts. Our data demonstrate partial compartmentalisation of cAMP signalling machinery in platelets, where lipid rafts and the actin cytoskeleton regulate the inhibitory effects induced by PGI_2. The increased platelet sensitivity to cAMP-elevating agents signalling upon raft and cytoskeleton disruption suggests that these compartments act to restrain basal cAMP signalling.     

Antiplatelet effect by p-cresol, a uremic and environmental toxicant, is related to inhibition of reactive oxygen species, ERK/p38 signaling and thromboxane A2 production

P-cresol is a well-known uremic toxin and environmental toxicant that may affect platelet functions. In this study, p-cresol (1-5 μM) inhibited the arachidonic acid (AA)-induced platelet aggregation, with 47% and 82% of inhibition at concentrations of 2 and 5 μM, respectively. Under similar experimental condition, p-cresol showed little effect on the U46619-induced platelet aggregation. p-cresol (<500 μM) revealed no discernable cytotoxicity to platelets as analyzed by quantification of lactate dehydrogenase release. Antiplatelet effect of p-cresol was related to inhibition of thromboxane A(2) (TXA(2)) and prostaglandin D(2) (PGD(2)) formation. P-cresol (2-100 μM) partly inhibited the AA-induced reactive oxygen species (ROS) production as well as the extracellular signal-regulated kinase (ERK1/2) and p38 phosphorylation in platelets. P-cresol further inhibited the AA-induced aggregation of rabbit platelet-rich plasma (PRP) with an IC50 of 2 μM and aggregation of human PRP (IC50 = 13.6 μM). Intravenous administration of p-cresol (250-1000 nmole) into mice effectively suppressed the ex vivo platelet aggregation, whereas showed little effect on the value of RBC, hemoglobin (HGB), hematocrit, MCV, MCH, MCHC, platelets and lymphocyte counts. These results indicate that in acute p-cresol-poisoning and long-term exposure to cresol as in severe uremic patients, p-cresol may potentially inhibit blood clot formation and lead to hemorrhagic disorders via inhibition of platelet aggregation, ROS production, ERK/p38 activation and TXA(2) production.